Optical approach for microfluidic DNA electrophoresis detection

ABSTRACT

Aspects of the disclosure provides a DNA analyzer to facilitate an integrated single-chip DNA analysis. The DNA analyzer includes an interface for coupling a microfluidic chip to the DNA analyzer. The microfluidic chip includes a first domain configured for polymerase chain reaction (PCR) amplification of DNA fragments, and a second domain fluidically coupled to the first domain to receive the DNA fragments and perform electrophoretic separation of the DNA fragments. The DNA fragments are tagged with fluorescent labels. The DNA analyzer includes a detection module to excite the fluorescent labels to emit fluorescence and detect the emitted fluorescence. The detection module includes a laser source, a set of optical elements, a filter module and a photo-detector.

INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No.12/659,492 filed Mar. 10, 2010, which claims the benefit of U.S.Provisional Applications No. 61/213,405, “Fast Sample to Answer DNAAnalyzer (Analytical Microdevice)” filed on Jun. 4, 2009, No.61/213,406, “Optical Approach for Microfluidic DNA ElectrophoresisDetection” filed on Jun. 4, 2009, and No. 61/213,404, “Multiple Sample,Integrated Microfluidic Chip for DNA Analysis” filed on Jun. 4, 2009,which are incorporated herein by reference in their entirety.

BACKGROUND

DNA is recognized as the “ultimate biometric” for human identification.DNA analysis can provide evidence for solving forensic and medicalcases, such as in areas of criminal justice, identifications of humanremains, paternity testing, pathogen detection, disease detection, andthe like.

SUMMARY

Aspects of the disclosure can provide a DNA analyzer to facilitate DNAanalysis. The DNA analyzer includes an interface for coupling amicrofluidic chip to the DNA analyzer. The microfluidic chip includes afirst domain configured for polymerase chain reaction (PCR)amplification of DNA fragments, and a second domain fluidically coupledto the first domain to receive the DNA fragments. The second domainincludes a separation channel for electrophoretic separation of the DNAfragments. The microfluidic chip may include other domains, such aspurification domain, post-PCR domain, and the like.

The DNA fragments are tagged with fluorescent labels during the PCRamplification. The DNA analyzer includes a detection module opticallycoupled with the microfluidic chip to excite the fluorescent labels toemit fluorescence and to detect the emitted fluorescence. The detectionmodule can include a laser source, a set of optical elements, a filtermodule and a photo-detector.

The laser source generates a laser beam. The set of optical elementsdirect the laser beam to the separation channel to excite thefluorescent labels to emit fluorescence while the DNA fragments migratein the separation channel. In addition, the set of optical elementscollect the emitted fluorescence into an optical signal. The filtermodule filters the optical signal to allow a first portion of theoptical signal having a first wavelength to pass, and the photo-detectorgenerates an electrical detection signal in response to the filteredoptical signal.

In an embodiment, the photo-detector includes a photo-multiplier tubeconfigured to generate the electrical detection signal in response tothe filtered optical signal. The set of optical elements include anobjective lens aligned with the separation channel to direct the laserbeam to the separation channel and to collect the emitted fluorescencefrom the separation channel. The objective lens can be aligned with theseparation channel by a motor.

The filter module can include an acousto-optic tunable filter (AOTF).The AOTF can filter the optical signal to allow the first portion of theoptical signal having the first wavelength to pass based on anelectrical tuning signal having a first tuning frequency. The firstwavelength satisfies a matching condition of the AOTF with the firsttuning frequency.

In an embodiment, the DNA analyzer includes a controller configured togenerate a control signal indicative of the first tuning frequency, anda synthesizer configured to generate the electrical tuning signal havingthe first tuning frequency based on the control signal.

The controller can adjust the control signal to be indicative of asecond tuning frequency. Then, the electrical tuning signal generated bythe synthesizer has the second tuning frequency. Based on the electricaltuning signal, the AOTF filters the optical signal to allow a secondportion of the optical signal having a second wavelength to pass. Thesecond wavelength satisfies the matching condition of the AOTF with thesecond tuning frequency.

In an embodiment, the DNA analyzer includes a modulation signalgenerator configured to generate a modulation signal having a modulationfrequency, and a reference signal having the modulation frequency. Themodulation signal being used by the AOTF to modulate the filteredoptical signal. Further, the DNA analyzer includes a phase-sensitivedetector configured to receive the reference signal and the electricaldetection signal corresponding to the modulated filtered optical signaland to demodulate the electrical detection signal based on the referencesignal.

It is noted that the DNA analyzer can include other modules to act onthe microfluidic chip to perform integrated single-chip DNA analysis.For example, the DNA analyzer can include a pressure module configuredto flow liquid in the microfluidic chip, a thermal module configured toinduce thermal cycling at the first domain of the microfluidic chip forthe PCR amplification, a power module configured to generate voltages tobe applied to the second domain of the microfluidic chip for theelectrophoretic separation, and a controller module. The controllermodule is configured to control the pressure module, the thermal module,the power module, and the detection module according to a controlprocedure to act on the microfluidic chip for a single-chip DNAanalysis.

Aspects of the disclosure can provide a method of DNA analysis. Themethod includes selecting a first wavelength corresponding to a firstfluorescent label used to label DNA fragments during polymerase chainreaction (PCR) amplification in a first domain of a microfluidic chip.The DNA fragments have been fluidically directed from the first domainto a second domain of the microfluidic chip having a separation channelfor electrophoretic separation. The method further includes exciting atleast the first fluorescent label to emit fluorescence in the seconddomain, and tuning a detection module to detect the emitted fluorescencehaving the first wavelength.

To excite the first fluorescent label to emit the fluorescence, themethod includes generating a laser beam, and directing the laser beam tothe separation channel to excite the first fluorescent label to emit thefluorescence while the DNA fragments migrate in the separation channel.The emitted fluorescence can be collected into an optical signal.

Further, to tune the detection module to detect the emitted fluorescencehaving the first wavelength, the method includes generating anelectrical tuning signal having a first tuning frequency, providing theelectrical tuning signal to an acousto-optic tunable filter (AOTF) inthe detection module to filter the optical signal and pass a firstportion of the optical signal having the first wavelength, and detectingthe filtered optical signal. The first wavelength satisfies a matchingcondition of the AOTF with the first tuning frequency.

In addition, the method includes selecting a second wavelengthcorresponding to a second fluorescent label used to label the DNAfragments during the (PCR) amplification in the first domain, andadjusting the electrical tuning signal to have a second tuningfrequency. The adjustment causes the AOTF to filter the optical signaland pass a second portion of the optical signal having the secondwavelength. The second wavelength satisfies the matching condition ofthe AOTF with the second tuning frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary embodiments of this disclosure will be described indetail with reference to the following figures, wherein like numeralsreference like elements, and wherein:

FIG. 1 shows a block diagram of an exemplary DNA analyzer according toan embodiment of the disclosure;

FIGS. 2A and 2B show a swab example and a sample cartridge exampleaccording to an embodiment of the disclosure;

FIG. 3 shows a schematic diagram of a microfluidic chip exampleaccording to an embodiment of the disclosure;

FIG. 4 shows a prototype implementation of a DNA analyzer according toan embodiment of the disclosure;

FIG. 5 shows a flow chart outlining a process example for using a DNAanalyzer to perform DNA analysis according to an embodiment of thedisclosure;

FIG. 6 shows a flow chart outlining a process example for a DNA analyzerto perform DNA analysis according to an embodiment of the disclosure;

FIG. 7 shows a block diagram of a detection module according to anembodiment of the disclosure;

FIG. 8 shows a block diagram of an optical design according to anembodiment of the disclosure;

FIG. 9 shows a block diagram for signal processing according to anembodiment of the disclosure; and

FIG. 10 shows a flow chart outlining a process example for a controllerto control a multi-color fluorescence detection according to anembodiment of the disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 shows a block diagram of an exemplary DNA analyzer 100 accordingto an embodiment of the disclosure. The DNA analyzer 100 includes amicrofluidic chip module 110, a thermal module 120, a pressure module130, a high voltage module 140, a detection module 150, a power module160, a computing module 170, and a controller module 180. Additionally,the DNA analyzer 100 can include a magnetic module 190. These elementscan be coupled together as shown in FIG. 1.

The DNA analyzer 100 is capable of processing sample-to-answer DNAanalysis on an integrated single-chip. Thus, using the DNA analyzer 100to perform DNA analysis does not need substantial experience andknowledge of DNA processes. In an example, the appropriate procedures touse the DNA analyzer 100 to perform DNA analysis can be learned in anhour. Additionally, the integrated single-chip DNA analysis requires areduced volume of reagents, for example, in the order of a micro-liter.Further, the reduced volume of reagents can reduce thermal inputs forinducing thermal cycles in the DNA analysis, and thus reduce the timefor DNA analysis.

The microfluidic chip module 110 includes a microfluidic chip 111. Themicrofluidic chip 111 can be suitably coupled with other elements of theDNA analyzer 100 to perform integrated single-chip DNA analysis. In anexample, the microfluidic chip module 110 is implemented as a disposablecartridge, and a cartridge interface that can couple the disposablecartridge with other components of the DNA analyzer 100 that are notincluded as part of the disposable cartridge. The disposable cartridgeincludes the microfluidic chip 111 and a micro-to-macro interface. Themicro-to-macro interface couples the microfluidic chip 111 to macrostructures on the disposable cartridge. The disposable cartridge can beseparately stored, and can be installed in the DNA analyzer 100 at atime of DNA analysis. After the DNA analysis, the disposable cartridgecan be suitably thrown away.

The microfluidic chip 111 includes various domains that can be suitablyconfigured to enable the integrated single-chip DNA analysis. In anembodiment, DNA analysis generally includes a step of PCR amplification,and a step of electrophoretic separation. The microfluidic chip 111 caninclude a first domain 111 a for the PCR amplification and a seconddomain 111 b for the electrophoretic separation. In addition, themicrofluidic chip 111 can include other domains that are suitablyintegrated with the first domain 111 a and the second domain 111 b. Inan example, the microfluidic chip 111 includes a purification domainfluidically coupled with the first domain 111 a. The purification domaincan be used to extract and purify a template DNA. It is noted that anysuitable techniques, such as solid-phase extraction, liquid-phaseextraction, and the like, can be used to purify the template DNA in thepurification domain.

In another example, the microfluidic chip 111 includes a post-PCRclean-up/dilution domain that is fluidically coupled with the firstdomain 111 a and the second domain 111 b. The post-PCR clean-up/dilutiondomain can be used for any suitable process after the PCR amplificationand before the electrophoretic separation.

The first domain 111 a includes a reservoir configured for PCRamplification. In an embodiment, the first domain 111 a includesmultiple separated reservoirs to enable simultaneous PCR amplificationfor multiple DNA samples. The temperature at the first domain 111 a canbe controlled by the thermal module 120 to enable the PCR amplification.According to an embodiment of the disclosure, the PCR amplification onthe microfluidic chip 111 requires only a small volume of reagents, andthe PCR amplification can achieve rapid thermal cycling. In an example,the volume of reagents used for the PCR amplification can be in theorder of sub-micro-liter, and the time required for the PCRamplification can be under 20 minutes.

The second domain 111 b can include a plurality of micro channels. Theplurality of micro channels can be configured for electrophoreticseparation. More specifically, each micro channel can be filled with,for example, polymer sieving matrix. Further, an electric field can beinduced in the micro channel. Thus, when DNA fragments are injected inthe micro channel, the DNA fragments can migrate by force of theelectric field at different speeds based on the sizes of the DNAfragments.

Additionally, the second domain 111 b can be configured to facilitateDNA fragments detection in the DNA analysis. In an example, DNAfragments are tagged with fluorescent labels during PCR, before beinginjected in the micro channels. The fluorescent labels can emitfluorescence of pre-known wavelength when excited by a laser beam. Thesecond domain 111 b includes a detection window configured fordetection. The laser beam can be directed to pass through the detectionwindow to excite the fluorescent labels in the micro channels. Theemitted fluorescence can pass through the detection window to becollected and detected.

The microfluidic chip 111 can include additional structures tofacilitate the integrated single-chip DNA analysis. For example, themicrofluidic chip 111 can include microfluidic channels that can directDNA fragments from the first domain 111 a to the second domain 111 b.Through the microfluidic channels, the DNA fragments flow in a solutionfrom the first domain 111 a to the second domain 111 b. In addition, themicrofluidic chip 111 can include inlets for receiving reagents and thetemplate DNA. The microfluidic chip 111 can also include additionalreservoirs for additional processing steps, such as dilution, cleanup,and the like.

The microfluidic chip 111 can be constructed from any suitable material.In an example, the microfluidic chip 111 is constructed from glass. Inanother example, the microfluidic chip 111 is constructed from plasticor polymeric material.

In addition to the microfluidic chip 111, the disposable cartridge caninclude a sample acceptor and a reagent carrier. In an example, thesample acceptor accepts a swab used for taking DNA sample, such as fromsaliva, bloodstains, cigarettes, and the like. Further, the sampleacceptor extracts a template DNA from the swab. The sample acceptor canuse any suitable mechanism, such as solid-phase extraction, liquid-phaseextraction, and the like to obtain and/or purify the template DNA fromthe swab. In an embodiment, the sample acceptor uses a solid-phase DNAextraction method, such as silica beads based DNA extraction.

In another embodiment, the sample acceptor uses a liquid-phase DNAextraction method. The liquid-phase DNA extraction method can simplifythe purification and extraction process, and reduce a total cost of theDNA analyzer 100. In an example, the sample acceptor uses an enzymaticDNA-isolation method to extract and purify the template DNA . Theenzymatic DNA-isolation method can achieve liquid phase purificationwithout a need of centrifugation. In addition, the sample acceptor canbe suitably designed to maintain sample integrity.

More specifically, the sample acceptor can include a plurality ofseparated wells for taking swabs, for example. Thus, the DNA analysiscan simultaneously process multiple DNA samples. Each well includes aliquid phase mixture that is sealed by a membrane at a bottom portion ofthe well. The liquid phase mixture can conduct enzymatic digestion ofall proteins and other cellular interferences, with the exception ofDNA. For example, the liquid phase mixture can include thermostableproteinases from thermophilic Bacillus species, such as disclosed inU.S. Patent Application Publication No. 2004/0197788, which isincorporated herein by reference in its entirety. Thus, the liquid phasemixture can perform DNA extraction and purification when a swab isimmersed in the liquid phase mixture. The liquid phase method canachieve comparable DNA quality to other methodologies in both DNAconcentration and purity. In an example, a final DNA concentration bythe liquid phase method is in a range of 0.5-2 ng/μL.

Further, using the liquid phase extraction method instead of the silicasolid phase method can reduce the overall hydraulic pressure requirementto induce solution flow through the microfluidic chip 111. In anembodiment, the liquid phase extraction can enable a valveless designfor the microfluidic chip 111. Thus, the liquid phase extraction cansimplify the DNA analyzer 100 and simplify the manufacturing and testingsteps in association with the solid-phase extraction.

Before taking DNA sample, a swab can be sealed in a hard case to avoidcontamination. The swab can be attached to a seal cap that can seal thehard case. The swab can be identified by various mechanisms. In anexample, a barcode label is attached to the hard case to identify theswab. In another example, the seal cap has a radio frequencyidentification (RFID) tag implanted. The RFID tag can identify the swabattached to the seal cap throughout the process. After the swab is usedto take DNA sample, the swab can be placed in one of the plurality ofseparated wells, and can be sealed in the well, for example, by the sealcap attached to the sampled swab. In an embodiment, the seal cap is astepped seal cap that can seal the well in a first step, and a secondstep. When the seal cap seals the well in the first step, the swab doesnot puncture the membrane. When the seal cap seals the well in thesecond step, the swab punctures the membrane and is immersed in theliquid phase mixture. The liquid phase mixture can then extract templateDNA from the swab.

The reagent carrier can house a plurality of reagents for DNA analysis,such as reagents for polymerase chain reaction (PCR) amplification,solutions for electrophoretic separation, and the like. In an STR typingexample, the reagent carrier houses reagents for multiplexed STRamplification. The reagents can perform multiplexed STR amplificationand can use multiple fluorescent dyes to label STR alleles. The reagentscan be commercially available reagent kits or can be tailored to themicro-scale chip environment to further facilitate the integratedsingle-chip DNA analysis.

In addition, the reagent carrier houses solutions that are suitable forelectrophoretic separation in the micro-scale chip environment. Forexample, the reagent carrier houses a coating solution, such aspoly-N-hydroxyethylacrylamide, and the like. The coating solution can beused to coat micro channel walls prior to the separation to reduceelectro osmotic flow and enable single base pair resolution of amplifiedDNA fragments. In another example, the reagent carrier houses a dilutionsolution, such as water and/or Formamide, and the like. The dilutionsolution can be used to reduce the ionic strength of the sample in orderto promote better electro-kinetic injection. In another example, thereagent carrier houses an internal lane standard (ILS). The ILS can beused for accurate size measurements. The reagent carrier also houses apolymer solution for electrophoretic separation in the micro-scale chipenvironment. The polymer solution is used as gels to provide a physicalseparation of DNA fragments according to chain length. For example, thepolymer solution can include a sieving or non-sieving matrix, such asthat disclosed in U.S. Pat. No. 7,531,073, No. 7,399,396, No. 7,371,533,No. 7,026,414, No. 6,811,977 and No. 6,455,682, which are incorporatedherein by reference in their entirety. In an example, a polymer sievingmatrix can be used to yield a single-base resolution in a totalseparation length of 8 cm and in less than 400 seconds.

The thermal module 120 receives control signals from the controllermodule 180, and induces suitable temperatures for DNA analysis, such asa temperature for DNA extraction, thermal cycles for the PCRamplification, a temperature for electrophoretic separation, and thelike. In an example, the thermal module 120 includes a resistance heaterto control a temperature in the wells of the sample acceptor for the DNAextraction and purification. In another example, the thermal module 120includes another resistance heater to control a temperature at thesecond domain 111 b.

In another example, the thermal module 120 includes a heating unit, acooling unit and a sensing unit to induce the thermal cycles for the PCRamplification at the first domain 111 a. The heating unit can directheat to the first domain 111 a, the cooling unit can disperse heat fromthe first domain 111 a, and the sensing unit can measure a temperatureat the first domain 111 a. The controller module 180 can control theheating unit and the cooling unit based on the temperature measured bythe sensing unit.

In an embodiment, the thermal module 120 performs non-contact thermalcontrols. For example, the thermal module 120 includes an infrared lightsource as the heating unit, a cooling fan as the cooling unit, and aninfrared pyrometer as the temperature sensing unit. The infrared lightsource, such as a halogen light bulb, can excite, for example, the 1.3μm vibrational band of liquid. Thus, the infrared light source can heata small volume of solution within a reservoir in the first domain 111 aindependent of the reservoir to achieve rapid heating and cooling. Theinfrared pyrometer measures blackbody radiation from an outside of thereservoir. In an example, the reservoir is designed to have a thinnerside for the infrared pyrometer measurements. The infrared pyrometermeasurements at the thinner side can more accurately reflect thetemperature of solution within the reservoir. Thus, the DNA analyzer 100can achieve a precise temperature control along with rapid thermalcycles. In an example, the DNA analyzer 100 can achieve a temperaturefluctuation of less than ±0.1° C., and a time of the thermal cycles forthe PCR amplification can be less than 20 minutes.

The pressure module 130 receives control signals from the controllermodule 180, and applies suitable pressures to the microfluidic chipmodule 110 to enable fluid movement. In an embodiment, the pressuremodule 130 receives a sensing signal that is indicative of a pressureapplied to the microfluidic chip module 110, and suitably adjusts itsoperation to maintain the suitable pressure to the microfluidic chipmodule 110.

The pressure module 130 can include a plurality of pumps. The pluralityof pumps control the injection of the various reagents and the templateDNA solutions into the microfluidic chip 111. According to an embodimentof the disclosure, the plurality of pumps can be individually controlledto achieve any possible timing sequence.

The pressure module 130 may include other pressure components to suitthe integrated single-chip integrated DNA analysis. In an embodiment,the microfluidic chip 111 has membrane valves. The pressure module 130can include a hydrodynamic pressure/vacuum system to suitably controlthe closing and opening of the membrane valves to enable fluid movementthrough the microfluidic chip 111.

In another embodiment, the microfluidic chip 111 is valveless. Forexample, the DNA analyzer 100 uses a liquid phase DNA extraction insteadof a silica solid phase DNA extraction. The liquid phase DNA extractioncan be integrated with following DNA processes on a valvelessmicrofluidic chip. Thus, the hydrodynamic pressure/vacuum system is notneeded. The pressure module 130 can be simplified to reduce thefootprint, the weight, the cost, and the complexity of the DNA analyzer100.

The power module 160 receives a main power, and generates variousoperation powers for various components of the DNA analyzer 100. In anexample, the DNA analyzer 100 is implemented using a modular design.Each module of the DNA analyzer 100 needs an operation power supply,which can be different from other modules. The power module 160 receivesan AC power input, such as 100-240 V, 50-60 Hz, single phase AC powerfrom a power outlet. Then, the power module 160 generates 5 V, 12 V, 24V, and the like, to provide operation powers for the various componentsof the DNA analyzer 100.

In addition, the power module 160 generates high voltages, such as 1000V, 2000 V, and the like, for suitable DNA processes on the microfluidicchip 111, such as electro-kinetic injection, electrophoretic separation,and the like.

Further, the power module 160 can implement various protectiontechniques, such as power outrage protection, graceful shut-down, andthe like, to protect the various components and data against powerfailure. It is noted that the power module 160 may include a back-uppower, such as a battery module, to support, for example, gracefulshut-down.

The high voltage module 140 can receive the high voltages from the powermodule 160 and suitably apply the high voltages on the microfluidic chip111. For example, the high voltage module 140 includes interfaces thatapply the high voltages to suitable electrodes on the microfluidic chip111 to induce electro-kinetic injection and/or electrophoreticseparation.

The detection module 150 includes components configured to suit theintegrated single-chip DNA analysis. In an embodiment, the detectionmodule 150 is configured for multicolor fluorescence detection. Thedetection module 150 includes a laser source unit, a set of optics and adetector unit.

The laser source unit emits a laser beam. In an example, the lasersource unit includes an argon-ion laser unit. In another example, thelaser source unit includes a solid state laser, such as a coherentsapphire optically pumped semiconductor laser unit. The solid statelaser has the advantages of reduced size, weight and power consumption.

The set of optics can direct the laser beam to pass through thedetection window at the second domain 111 b of the microfluidic chip111. The laser beam can excite fluorescent labels attached to DNAfragments to emit fluorescence. Further, the set of optics can collectand direct the emitted fluorescence to the detector unit for detection.In an STR typing example, STR alleles are separated in the second domain111 b according to sizes. STR alleles of different sizes pass thedetection window at different times. In addition, STR alleles ofoverlapping sizes can be tagged with fluorescent labels of differentcolors. The detector unit can be configured to detect an STR allelehaving a fluorescent label based on a time of fluorescence emitted bythe fluorescent label and a color of the emitted fluorescence.

In another example, internal lane standard (ILS) is added to migrate inthe micro channel with the STR alleles. The ILS includes DNA fragmentsof known sizes, and can be tagged with a pre-determined fluorescent dye.The detector unit detects fluorescence emitted from the ILS to set up asize scale. In addition, the detector unit detects fluorescence emittedfrom the STR alleles. The detector unit can suitably convert thedetected fluorescence into electrical signals. The electrical signalscan be suitably stored and/or analyzed. In an example, a processorexecutes DNA analysis software instructions to identify the STR allelesby their sizes and emitted fluorescence colors (wavelengths).

The computing module 170 includes computing and communication units. Inan example, the computing module 170 includes a personal computer. Thepersonal computer can be coupled with the controller module 180 toprovide a user interface. The user interface can inform the status ofthe DNA analyzer 100, and can receive user instructions for controllingthe operation of the DNA analyzer 100. The personal computer includesvarious storage media to store software instruction and data. Thepersonal computer can include DNA analysis software that can performdata processing based on raw data obtained from the detection module150. In addition, the personal computer can be coupled to externalprocessing units, such as a database, a server, and the like to furtherprocess the data obtained from the DNA analyzer 100.

The magnetic module 190 can enable a magnetic solid phase for theintegrated single chip DNA analysis. In an embodiment, the magneticsolid phase can be suitably incorporated in the integrated single chipDNA analysis to facilitate a volume reduction to suit for low copynumbers of template DNAs. In another embodiment, the magnetic solidphase can be suitably incorporated into an integrated single chipsequencing DNA analysis.

The controller module 180 can receive status signals and feedbacksignals from the various components, and provide control signals to thevarious components according to a control procedure. In addition, thecontroller module 180 can provide the status signals to, for example,the personal computer, to inform the user. Further, the controllermodule 180 can receive user instructions from the personal computer, andmay provide the control signals to the various components based on theuser instructions.

During operation, the controller module 180 receives user instructionsfrom the personal computer to perform a STR typing analysis, forexample. The controller module 180 then monitors the microfluidic chipmodule 110 to check whether a suitable disposable cartridge has beeninstalled, and whether swabs have been identified and suitably immersedin the liquid phase mixture to extract template DNA. When the controllermodule 180 confirms the proper status at the microfluidic chip module110, the controller module 180 starts a control procedure correspondingto the STR typing analysis. In an example, the controller module 180 cancontrol the thermal module 120 to maintain an appropriate temperature atthe wells of the sample acceptor for a predetermined time. The liquidphase mixture in the wells can extract template DNAs from the swabs.Then, the controller module 180 can control the pressure module 130 topump the extracted template DNAs into the first domain 111 a of themicrofluidic chip 111. In addition, the controller module 180 cancontrol the pressure module 130 to pump reagents for multiplexed STRamplification into the first domain 111 a.

Further, the controller module 180 can control the thermal module 120 toinduce thermal cycling for the multiplexed STR amplification at thefirst domain 111 a. The reagents and the thermal cycling can cause DNAamplification. In addition, the DNA amplicons can be suitably taggedwith fluorescent labels.

Subsequently, the controller module 180 can control the pressure module130 to flow the DNA amplicons to the second domain 111 b. The controllermodule 180 may control the pressure module 130 to pump a dilutionsolution into the microfluidic chip 111 to mix with the DNA amplicons.In addition, the controller module 180 may control the pressure module130 to pump an ILS into the microfluidic chip 111 to mix with the DNAamplicons.

Further, the controller module 180 controls the high voltage module 140to induce electro-kinetic injection to inject DNA fragments into themicro channels. The DNA fragments include the amplified targets, and theILS. Then, the controller module 180 controls the high voltage module140 to induce electrophoretic separation in the micro channels.Additionally, the controller module 180 can control the thermal module120 to maintain a suitable temperature at the second domain 111 b duringseparation, for example, to maintain the temperature for denaturingseparation of the DNA fragments.

The controller module 180 then controls the detection module 150 todetect the labeled DNA fragments. The detection module 150 can emit anddirect a laser beam to the micro channels to excite the fluorescentlabels to emit fluorescence. Further, the detection module 150 candetect the emitted fluorescence and store detection data in a memory.The detection data can include a detection time, and a detected color(wavelength), along with a detected intensity, such as a relativemagnitude of the detected fluorescence. The detection data can betransmitted to the personal computer for storage. Additionally, thecontroller module 180 can provide control statuses to the personalcomputer to inform the user. For example, the controller module 180 cansend an analysis completed status to the personal computer when thecontrol procedure is completed.

The DNA analyzer 100 can be suitably configured for various DNA analysesby suitably adjusting the reagents housed by the reagent carrier and thecontrol procedure executed by the controller module 180.

FIG. 2A shows a swab storage example 212, and FIGS. 2B-2C show a sideelevation view and a front elevation view of a sample cartridge example215 according to an embodiment of the disclosure. The swab storage 212includes a case 203, a seal cap 202 and a swab 205. The seal cap 202 andthe swab 205 are attached together. In addition, the swab storage 212includes an identifier, such as a barcode label 204 that can be attachedto the case 203, an RFID tag 201 that can be implanted in the seal cap202, and the like.

Before taking DNA sample, the swab 205 is safely stored in the case 203to avoid contamination. After taking DNA sample, the swab 205 can beplaced in the sample cartridge 215.

The sample cartridge 215 can include a microfluidic chip 211, a sampleacceptor 207 and a reagent carrier 206. The sample acceptor 207 includesa plurality of separated wells 207A-207D for taking swabs. Each wellincludes a liquid phase mixture 214 that is sealed by a membrane 208 ata bottom portion of the well. The liquid phase mixture 214 can conductenzymatic digestion of all proteins and other cellular interferences,with the exception of DNA, and thus can perform DNA extraction andpurification when a swab with DNA sample is inserted in the liquid phasemixture 214.

While the sample cartridge 215 is described in the context of swabs, itshould be understood that the sample cartridge 215 can be suitablyadjusted to suit other DNA gathering methods, such as blood stain cards,airborne samples, fingerprints samples, and the like.

In an embodiment, the seal cap 202 is a stepped seal cap that can sealthe well in a first step, and a second step. When the seal cap 202 sealsthe well in the first step, the swab 205 does not puncture the membrane208, and can be safely sealed in the well to maintain sample integrity.When the seal cap 202 seals the well in the second step, the swab 205punctures the membrane 208 and is immersed in the liquid phase mixture214.

The reagent carrier 206 houses various solutions for DNA analysis. In anSTR typing example, the reagent carrier houses reagents for multiplexedSTR amplification. In addition, the reagent carrier houses a coatingsolution, such as poly-N-hydroxyethylacrylamide, and the like. Thecoating solution can be used to coat micro channel walls prior to theseparation. Further, the reagent carrier houses a dilution solution,such as water, formamide, and the like. The dilution solution can beused to reduce the ionic strength in order to promote betterelectro-kinetic injection. In an embodiment, the reagent carrier housesan internal lane standard (ILS). The ILS can be used for sizemeasurement. The reagent carrier also houses a polymer solution forelectrophoretic separation in the micro-scale chip environment.

During operation, for example, a new disposable cartridge 215 is takenfrom a storage package, and installed in a DNA analyzer, such as the DNAanalyzer 100. Then, a swab 205 can be used to take a DNA sample. Theswab 205 is then identified and inserted into one of the wells 207A-207Dand sealed in the first step. Additional swabs 205 can be used to takeDNA samples, and then identified and inserted into the un-used wells207A-207D. Further, the DNA analyzer 100 can include a mechanism thatcan push the seal caps 202 to seal the wells 207A-207D in the secondstep, thus the swabs 205 can puncture the membrane 208, and immerse inthe liquid phase mixture 214.

FIG. 3 shows a schematic diagram of a microfluidic chip example 311according to an embodiment of the disclosure. The microfluidic chip 311includes various micro structures, such as inlets 312-314, reactionreservoirs 315-316, channels 317 a-317 b, electrode reservoirs 318,outlets (not shown), and the like, that are integrated for single-chipDNA analysis. It is noted that the various micro structures can bedesigned and integrated to suit for various DNA analyses, such as STRtyping, sequencing, and the like.

The inlets 312-314 can be coupled to a pressure module to injectsolutions in the microfluidic chip 311. As described above, theconnection can be made via a micro-macro interface. In an example, theinlet 312 is for injecting a template DNA solution from a well of thesample acceptor 207, and the inlet 313 is for injecting PCR reagentsfrom the reagent carrier 206. In addition, the inlet 313 can be used forinjecting dilution solution and ILS from the reagent carrier 206.

The reaction reservoirs 315-316 are configured for various purposes. Inan example, the reaction reservoir 315 is configured for the PCRamplification, and the reaction reservoir 316 is configured for thepost-PCR processes, such as dilution, and the like. More specifically,the reaction reservoir 315 is located in a first domain 311 a, which isa thermal control domain. The temperature within the thermal controldomain 311 a can be precisely controlled. In an example, an infraredheating unit directs heat to the thermal control domain 311 a, a coolingfan disperses heat from the thermal control domain 311 a, and aninfrared sensing unit measures a temperature in the thermal controldomain 311 a. The infrared heating unit and the cooling fan can becontrolled based on the temperature measured by the infrared sensingunit. The infrared heating unit, the cooling fan, and the infraredsensing unit can perform thermal control without contacting the thermalcontrol domain 311 a.

In another example, the temperature in the thermal control domain 311 ais measured by a thermal coupling technique. More specifically, themicrofluidic chip 311 includes a thermal-coupler reservoir 319 withinthe first domain 311 a. Thus, the solution temperature within thereaction reservoir 315 and the thermal-coupler reservoir 319 can beclosely related. The solution temperature within the thermal-couplerreservoir 319 can be measured by any suitable technique. Based on themeasured solution temperature within the thermal-coupler reservoir 319,the solution temperature within the reaction reservoir 315 can bedetermined. Then, the infrared heating unit and the cooling fan can becontrolled based on the temperature measured by the thermal couplingtechnique in order to control the solution temperature in the reactionreservoir 315.

In an embodiment, after the PCR amplification, the PCR mixture isfluidically directed from the reaction reservoir 315 to a post-PCRclean-up/dilution domain, such as the reaction reservoir 316. In thereaction reservoir 316, the PCR mixture is diluted. In an example, thePCR mixture and a dilutant solution are mixed together according to aratio from 1:5 to 1:20 (1 part of PCR mixture to 5-20 parts ofdilutant). Further, ILS can be added in the reaction reservoir 316 tomix with the PCR mixture.

The channels 317 a-317 b are located in a second domain 311 b. Electricfields can be suitably applied onto the channels 317 a-317 b. In anexample, the channels 317 a-317 b are configured according to a cross-Tdesign, having a short channel 317 a and a long channel 317 b.

The electrode reservoirs 318 can be used to apply suitable electricfields over the short channel 317 a and the long channel 317 b. Thus,the short channel 317 a is configured for electro-kinetic injection, andthe long channel 317 b is configured for electrophoretic separation. Forexample, when a high voltage is applied to the short channel 317 a, DNAfragments can be injected from the reaction reservoir 316 into the shortchannel 317 a at the intersection of the short channel 317 a and thelong channel 317 b. The long channel 317 b can be filed with sievingmatrix. When a high voltage is applied to the long channel 317 b, theinjected DNA fragments can migrate in the long channel 317 b to thepositive side of the electric field induced by the high voltage, in thepresence of the sieving matrix. In an example, the length of the longchannel 317 b is about 8.8 cm with detection at about 8 cm from theintersection.

It should be understood that the microfluidic chip 311 can include otherstructures to assist DNA analysis. In an example, the microfluidic chip311 includes an alignment mark 321. The alignment mark 321 can assist adetection module to align to the long channel 317 b.

During operation, for example, the inlet 312 can input a template DNAinto the reaction reservoir 315, and the inlet 313 can input PCRreagents into the reaction reservoir 315. Then, thermal-cycling can beinduced at the first domain 311 a, and PCR amplification can beconducted in the reaction reservoir 315 to amplify DNA fragments basedon the template DNA and the PCR reagents. After the PCR amplification,the DNA amplicons in the reaction reservoir 315 can be mobilized intothe reaction reservoir 316 in a liquid flow. In the reaction reservoir316, a dilution solution and ILS can be input to mix with the DNAfragments. Further, the DNA fragments in the reaction reservoir 316 canbe injected across the short channel 317 a by electro-kinetic injection.The DNA fragments then migrate in the long channel 317 b under the forceof electric field applied over the long channel 317 b. The speed ofmigration depends on the sizes of the DNA amplicons, in the presence ofthe sieving matrix. Thus, the DNA fragments are separated in the longchannel 317 b according to their sizes.

FIG. 4 shows an exemplary DNA analyzer 400 according to an embodiment ofthe disclosure. The DNA analyzer 400 is packaged in a box. The boxincludes handles, wheels and the like, to facilitate transportation ofthe DNA analyzer 400. In an implementation, the total weight of the DNAanalyzer 400 is less than 70 lb, and is appropriate for two persons tocarry.

The DNA analyzer 400 is implemented in a modular manner. Each module canbe individually packaged, and can include an interface for inter-modulecouplings. Thus, each module can be easily removed and replaced. Themodular design can facilitate assembly, troubleshooting, repair, and thelike.

The DNA analyzer 400 includes a user module (UM) 410, an active pressuremodule (APM) 430, a detection module 450, a power module (PM) 460, acomputing module 470, and a controller module (CM) 480. In addition, theDNA analyzer 400 includes a sample cartridge storage 415 and a swabstorage 412.

The UM 410 includes a holder to hold a sample cartridge, such as thesample cartridge 215, at an appropriate position when the samplecartridge is inserted by a user. Further, the UM 410 includes interfacecomponents to couple the sample cartridge 215 with, for example, the APM430, the detection module 450, and the like. The UM 410 includes thermalcomponents, such as resistance heaters 421, a cooling fan 422, aninfrared heating unit 423, and the like. The thermal components can besuitably positioned corresponding to the sample cartridge 215. Forexample, a resistance heater 421 is situated at a position that caneffectively control a temperature of the liquid phase mixture within theplurality of separated wells on the sample cartridge 215. Thetemperature can be determined to optimize enzyme activities of theliquid phase mixture to conduct enzymatic digestion of all proteins andother cellular interferences, with the exception of DNA. Anotherresistance heater 421 is at a position that can effectively control atemperature of the separation channel on the microfluidic chip 211. Theinfrared heating unit is at a position that can direct heat to thethermal control domain of the microfluidic chip 211 on the samplecartridge 215. The cooling fan is at a position that can effectivelydisperse heat from the thermal control domain. Further, the UM 410includes a high voltage module that can apply suitable high voltages viathe electrode reservoirs of the microfluidic chip 211.

It is noted that the UM 410 can include other suitable components. In anembodiment, the UM 410 includes a magnetic module that can suitablyapply magnetic control over a domain of the microfluidic chip 211.

The APM 430 includes suitably components, such as pumps, vacuums, andthe like, to apply suitable pressures to the microfluidic chip 211 toenable fluid movement.

The PM 460 receives an input main power, and generates various operationpowers, such as 6 V, 12 V, 24 V, 1000V, 2000V, and the like, for variouscomponents of the DNA analyzer 400.

The detection module 450 can include a laser module (LM) 451, a passiveoptics module (POM) 452, and an active optics module (AOM) 453. The LM451 can include any suitable device to emit a laser beam. In anembodiment, the LM 451 includes an argon-ion laser. In another example,the LM 451 includes a diode laser. In another embodiment, the LM 451includes a solid state laser, such as a coherent sapphire opticallypumped semiconductor laser. The solid state laser can have a reducedsize and weight, and can consume less power than the argon-ion laser. Inaddition, the solid state laser generates less waste heat, such that fansize can be reduced to reduce footprint of the DNA analyzer 400.

The AOM 453 includes optical elements that may need to be adjusted withregard to each inserted microfluidic chip. In an example, the AOM 453includes a plurality of optical fibers that are respectively coupled toa plurality of separation channels on the microfluidic chip. Theplurality of optical fibers can respectively provide laser beams to theplurality of separation channels to excite fluorescence emission. Inaddition, the plurality of optical fibers can return the emittedfluorescence from the plurality of separation channels.

The POM 452 includes various optical elements, such as lens, splitters,photo-detectors, and the like, that do not need to be adjusted withregard to each inserted microfluidic chip. In an example, the POM 452 iscalibrated and adjusted with regard to the LM 451 and the AOM 453 whenthe detection module 450 is assembled. Then, the optical elements withinthe POM 452 are situated at relatively fixed positions, and do not needto be adjusted with regard to each inserted microfluidic chip.

The controller module 480 is coupled to the various components of theDNA analyzer 400 to provide control signals for DNA analysis. Thecontroller module 480 includes a control procedure that determinessequences and timings of the control signals.

The computing module 470 is implemented as a personal computer. Thepersonal computer includes a processor, a memory storing suitablesoftware, a keyboard, a display, and a communication interface. Thecomputing module 470 can provide a user interface to ease user controland monitor of the DNA analysis by the DNA analyzer 400.

FIG. 5 shows a flow chart outlining a process example for using a DNAanalyzer, such as the DNA analyzer 400, to perform DNA analysisaccording to an embodiment of the disclosure. The process starts atS501, and proceeds to S510.

At S510, a user of the DNA analyzer 400 plugs in a main power supply. Inan embodiment, the main power supply can be a 110 V, 50 Hz, AC powersupply, or can be a 220V, 60 Hz, AC power supply. The power module 460can convert the main power supply to a plurality of operation powers,and provide the plurality of operation powers to the various modules ofthe DNA analyzer 400. Then, the process proceeds to S515.

At S515, the user starts up a user control interface. For example, theuser turns on the personal computer 470, and starts a software packagethat interacts with the user and the controller module 480. The softwarepackage enables the personal computer 470 to provide a user controlinterface on the display. Further, the software package enables thepersonal computer 470 to receive user instructions via the keyboard ormouse. The software packages can also enable the personal computer 470to communicate with the controller module 480. Then, the processproceeds to S520.

At S520, the user instructs the DNA analyzer 400 to initialize. The usercontrol interface receives the initialization instruction, and thesoftware package enables the personal computer 470 to send theinitialization instruction to the controller module 480. The controllermodule 480 can then initialize the various components of the DNAanalyzer 400. For example, the controller module 480 can power on thevarious components, check the status and reset the status if needed.Then, the process proceeds to S525.

At S525, the user inserts a sample cartridge 215 in the UM 410. Thesample cartridge 215 can be positioned by a holder. The interfacecomponents can suitably couple the sample cartridge 215 to othercomponents of the DNA analyzer 400. Then, the process proceeds to S530.

At S530, the user takes a swab 205, and lets the DNA analyzer 400 toidentify the swab 205. In an example, the DNA analyzer 400 includes abarcode reader that can read the barcode label 204 attached to the case203 for storing the swab 205. In another example, the DNA analyzer 400excites the RFID 201 implanted in the seal cap 202 of the swab 205 toobtain a unique serial number of the swab 205. Then, the processproceeds to S535.

At S535, the user uses the swab 205 to take a DNA sample and inserts theswab 205 into a well of the sample cartridge 215. The user may repeatthe steps S530 and S535 to insert multiple swabs 205 into the separatedwells of the sample cartridge 215. Then, the process proceeds to S540.

At S540, the user instructs the DNA analyzer 400 to start a DNAanalysis. The user control interface receives the start instruction, andthe software package enables the personal computer 470 to send the startinstruction to the controller module 480. The controller module 480 canstart a control procedure corresponding to the DNA analysis. In anexample, the controller module 480 starts an STR typing procedurecorresponding to a multiplexed STR typing analysis. In another example,the controller module 480 starts a sequencing procedure corresponding toDNA sequencing analysis. Then, the process proceeds to S545.

At S545, the user waits and monitors the status of the DNA analysis. Thecontrol procedure can specify sequences and timings of control signalsto various components of the DNA analyzer 400 corresponding to the DNAanalysis. Then, the controller module 480 automatically sends thecontrol signals according to the sequences and the timings specified inthe control procedure. In addition, the controller module 480 receivesstatus and feedback signals from the various components, and sends themto the personal computer 470. The personal computer 470 then providesthe analysis status for the user to monitor. Then, the process proceedsto S550.

At S550, the controller module 480 finishes executing the controlprocedure, and sends an analysis-completed status to the personalcomputer 470. The personal computer 470 can inform the user of theanalysis-completed status via the user control interface. Then, theprocess proceeds to S555.

At S555, the user performs post data processing. The user can store theraw data of the DNA analysis, or transmit the raw data to a remotereceiver. In addition, the user may start a software package for postdata processing. Alternatively, the software package for post dataprocessing can be suitably integrated with the control procedure. Thus,after the control procedure is successfully executed, the softwarepackage for post data processing is executed automatically to performpost data processing. The process then proceeds to S599 and terminates.

It is noted that to perform another DNA analysis, the user may throwaway the sample cartridge and repeat S520-S550. It is also noted thatthe sequence of the DNA analysis steps can be suitably adjusted. Forexample, S535 and S530 can be swapped, thus a swab can be first used totake a DNA sample, and then identified by the DNA analyzer 400.

FIG. 6 shows a flow chart outlining a process example 600 for a DNAanalyzer to perform multiplexed STR typing according to an embodiment ofthe disclosure. The process starts at S601 and proceeds to S610.

At S610, the controller module 480 controls the resistance heater 421 tomaintain a temperature for template DNA extraction and purification.More specifically, the resistance heater 421 is positioned correspondingto the plurality of wells on the sample cartridge 215. A well can accepta swab 205. The swab 205 can puncture the membrane that seals the liquidphase mixture at the bottom of the well, thus the swab 205 is immersedinto the liquid phase mixture. The liquid phase mixture can extract andpurify a template DNA from the swab at the temperature according toenzymatic DNA isolation method. In an embodiment, the liquid phasemixture can achieve a compatible DNA concentration and purity to silicabased solid phase extraction method in about 6 minutes. Then, theprocess proceeds to S620.

At S620, the controller module 480 controls the APM 430 to flow theextracted template DNA and reagents to a reaction reservoir for the PCRamplification. For example, the reagent carrier 206 houses reagents formultiplexed STR amplification. The controller module 480 sends controlsignals to the APM 430. In response to the control signals, a pump pumpsthe liquid phase mixture from the well to the reaction reservoir, andanother pump pumps the reagents from the reagent carrier 206 to thereaction reservoir. Then, the process proceeds to S630.

At S630, the controller module 480 controls the cooling fan 422 and theinfrared heating unit 423 to induce thermal cycling in the reactionreservoir for the multiplexed STR amplification. In addition, thereagents can attach fluorescent labels to the DNA amplicons during theSTR amplification process. The process then proceeds to S640.

At S640, after the PCR amplification, the solution can be diluted. Morespecifically, the controller module 480 sends control signals to the APM430 after the PCR amplification. In response to the control signals, theAPM 430 flows the DNA amplicons into a dilution reservoir. In addition,the APM 430 flows a dilution solution from the reagent carrier into thedilution reservoir. The process then proceeds to S650.

At S650, the controller module 480 sends control signals to the highvoltage module in the UM 410 to inject the DNA amplicons across theinjection arm (the short channel 317 a). Then, the process proceeds toS660.

At S660, the controller module 480 sends control signals to the highvoltage module in the UM 410 to apply appropriate high voltage over theseparation channel (the long channel 317 b) to separate the DNAamplicons based on sizes. The process then proceeds to S670.

At S670, the controller module 480 sends control signals to thedetection module 450 to excite the fluorescent labels to emitfluorescence and detect the emitted fluorescence. The raw detection datacan be sent to the personal computer 470 for storage andpost-processing. The process then proceeds to S699, and terminates.

It is noted that some process steps in the process 600 can be executedin parallel. For example, the step S660 and the step S670 can beexecuted in parallel. The controller module 480 sends control signals toboth the high voltage module in the UM 410 and the detection module 450at about the same time. The control signals to the high voltage modulein the UM 410 cause the electrophoretic separation in the separationchannel, while the control signals to the detection module 450 causefluorescence detection.

It is noted that the process 600 can be suitably adjusted along withreagents adjustments for other DNA analysis, such as qPCR DNAquantitation, sequencing, and the like.

In a qPCR DNA quantitation example, step S601 to S630 are executed, andstep S640 to S670 can be deleted. In addition, in step S630, whenthermal cycles are induced in a qPCR reservoir for PCR amplification,the controller module 480 sends control signals to the detection module450 to detect florescence emitted by the fluorescent labels in the qPCRreservoir.

It is also noted that a magnetic solid phase purification process stepcan be suitably added into the process 600 to facilitate further volumereduction, thus the process 600 can be adjusted for DNA sequencing.

FIG. 7 shows a block diagram of an exemplary detection module 750coupled with an exemplary sample cartridge 715 having a microfluidicchip 711 according to an embodiment of the disclosure. The detectionmodule 750 can be suitably installed in a DNA analyzer, such as the DNAanalyzer 100, or the DNA analyzer 400. Further, the detection module 750can be coupled with other components, such as a controller module of theDNA analyzer. The controller module can control the detection module750, and other modules, such as thermal module, pressure module, highvoltage module, and the like, to act on the microfluidic chip 711 toperform an integrated single-chip DNA analysis. The detection module 750includes a laser module 751, a passive optics module 752 and an activeoptics module 753. These elements can be coupled together as shown inFIG. 7.

The microfluidic chip 711 can be configured for an integratedsingle-chip DNA analysis, such as the microfluidic chip 311 shown inFIG. 3. The microfluidic chip 711 includes various domains that can besuitably configured for various purposes. For example, the microfluidicchip 711 includes a first domain configured for PCR amplification and asecond domain having a separation channel configured for electrophoreticseparation. Additionally, the microfluidic chip 711 includes, forexample, purification domain, post-PCR clean-up/dilution domain, and thelike.

The detection module 750 is optically coupled to the microfluidic chip711. As described above, the microfluidic chip 711 includes a separationchannel configured for electrophoretic separation of DNA fragments. TheDNA fragments migrate in the separation channel based on their sizes.The DNA fragments can be suitably tagged with fluorescent labels. Thefluorescent labels can be optically detected by the detection module750. Based on the detected fluorescent labels, DNA analyses, such asidentification, sequencing, and the like, can be suitably performed.

More specifically, the detection module 750 directs a laser beam to alocation of the separation channel along the migration direction of theDNA fragments. The laser beam can excite the fluorescent labels attachedto the DNA fragments to emit fluorescence when the DNA fragments migratethrough the location. The detection module 750 collects the emittedfluorescence and detect properties of the fluorescence, such asintensity, wavelength, timing, and the like. The detected properties canbe suitably stored, and analyzed.

The laser module 751 can include any suitably laser device, such as anargon-ion laser device, a solid state laser, and the like, to generatethe laser beam. In an example, the laser module 751 includes a coherentsapphire optically pumped semiconductor laser (OPSL) outputs a laserbeam of 488 nm wavelength, and has an output power of 200 mW. The lasermodule 751 provides the laser beam to the passive optics module 752 viaany suitable optical channel, such as an optical fiber, and the like.

The passive optics module 752 interfaces with the active optics module753 and the laser module 751. The passive optics module 752 receives thelaser beam from the laser module 751 and transmits the laser beam to theactive optics module 753. On the other side, the passive optics module752 receives an optical signal returned by the active optics module 753.Further, the passive optics module 752 converts the optical signal intoan electrical signal, and suitably processes the electrical signal.

The passive optics module 752 includes various optical components, suchas a set of optics 790 and a photo-detector 799, that are generallysituated at substantially fixed positions. In an example, the opticalcomponents within the passive optics module 752 are pre-calibrated andfixed at their calibrated positions by the manufacture. In anotherexample, the optics components are calibrated with regard to the activeoptics module 753 and the laser module 751 when the detection module 750is assembled together. Then, the optical components are situated attheir calibrated positions, and do not need to be adjusted for everysample cartridge 715. It is noted that the passive optics module 752 mayadjust the optical components, for example, during a maintenanceprocedure.

The active optics module 753 receives the laser beam from the passiveoptics module 752, and suitably directs the laser beam to the separationchannel on the microfluidic chip 711. On the other hand, the activeoptics module 753 collects fluorescence emitted by the fluorescentlabels into an optical signal, and transmits the optical signal to thepassive optics module 752.

The active optics module 753 includes optical components that may needto be adjusted for each sample cartridge 715. In the FIG. 7 example, theactive optics module 753 includes a motor 756 coupled to an objectivelens 791. The motor 756 can adjust the objective lens 791 to focus thelaser beam onto a location of the separation channel on the samplecartridge 715.

The detection module 750 is implemented in a modular manner. Each of thelaser module 751, the passive optics module 752 and the active opticsmodule 753 can be individually handled, such as manufactured, purchased,tested, and calibrated. Further, the laser module 751, the passiveoptics module 752 and the active optics module 753 can be suitablycoupled together, and assembled in a DNA analyzer. During operation, theactive optics module 753 can be calibrated with regard to themicrofluidic chip 711 on the sample cartridge 715. The laser module 751and the passive optics module 752 do not need to be adjusted for everysample cartridge 715.

During operation, for example, when a new sample cartridge 715 isinstalled in a DNA analyzer having the detection module 750, the DNAanalyzer can start an initialization process to calibrate the detectionmodule 750 with regard to a microfluidic chip 711 on the samplecartridge 715. During the initialization process, the motor 756 alignsthe objective lens 791 to a separation channel on the microfluidic chip711. In an example, the microfluidic chip 711 includes an alignment markto assist the active optics module 753 to align the objective lens 791to a desired location on the separation channel.

Further, the DNA analyzer starts a control procedure to control thevarious components of the DNA analyzer to act on the microfluidic chip711 in order to perform an integrated single-chip DNA analysis. Forexample, template DNA can be suitably extracted and fluidically directedto the first domain of the microfluidic chip 711; a PCR amplificationcan be suitably induced in the first domain of the microfluidic chip 711to amplify DNA fragments; then the amplified DNA fragments are suitablyinjected into the separation channel of the microfluidic chip 711; andthen electrophoretic separation can be suitably induced in theseparation channel. In addition, the detection module 750 can becontrolled to direct a laser beam to the separation channel to excitefluorescent labels used to tag the DNA fragments. The fluorescent labelsemit fluorescence. The detection module 750 collects the fluorescenceinto an optical signal, returns the optical signal, and detectsfluorescence information in the optical signal. The detectedfluorescence information can be suitably stored, and further processedby the DNA analyzer, or can be transmitted to other device for furtherprocessing.

FIG. 8 shows a block diagram of an optics module 852 coupled with amicrofluidic chip 811 and a laser module 851 according to an embodimentof the disclosure. The optics module 852 includes an objective lens 891,a dichroic mirror 892, a long pass filter 889, a front surface mirror893, a pinhole 894, a first acromat lens unit 895, an acousto-optictunable filter (AOTF) 896, a beam block 897, a second acromate lens unit898, and a photomultiplier tube (PMT) 899. These elements can besuitably coupled together as shown in FIG. 8.

The laser module 851 emits a laser beam. The laser beam is directed to aseparation channel on the microfluidic chip 811 via a first path P1formed by the elements of the optics module 852. The laser beam canexcite fluorescent labels in the separation channel to emitfluorescence. The emitted fluorescence is collected into an opticalsignal, and suitably returned to the PMT 899 via a second path P2 formedby the elements of the optics module 852.

The first path P1 includes the dichroic mirror 892 and the objectivelens 891. The dichroic mirror 892 is configured to reflect light orallow light to pass through based on wavelength. In an example, thedichroic mirror 892 is configured to reflect light when the wavelengthof the light is about 488 nm, and allow light to pass through when thewavelength of the light is larger than 525 nm. Thus, when the lasermodule 851 is configured to generate the laser beam having a wavelengthof 488 nm and the laser beam is suitably directed to the dichroic mirror892, the dichroic mirror 892 reflects the laser beam. The reflectedlaser beam is directed to the objective lens 891. The objective lens 891focuses the laser beam to the separation channel on the microfluidicchip 811. In an embodiment, the objective lens 891 is coupled with amotor (not shown). The motor is used to adjust the objective lens 891 tofocus the laser beam to the separation channel on the microfluidic chip811.

The second path P2 includes the objective lens 891, the dichroic mirror892, the long pass filter 889, the front surface mirror 893, the pinhole894, the first acromat lens unit 895, the AOTF 896, the beam block 897,the second acromat lens unit 898, and the PMT 899.

The objective lens 891 collects the fluorescence emitted by thefluorescent labels to form an optical signal, and return the opticalsignal to the dichroic mirror 892. The fluorescent labels can besuitably selected, such that the wavelength of the emitted fluorescenceis larger than 525 nm. Thus, the dichroic mirror 892 allows thefluorescence emitted by the fluorescent labels to pass through, anddirects the passed optical signal to the long pass filter 889. The longpass filter 889 further filters the optical signal. More specifically,the long pass filter 889 can be suitably configured to allow the emittedfluorescence to pass through, and filter out shorter wavelengths fromthe optical signal.

The front surface mirror 893 is used to change the direction of theoptical signal, and thus directs the optical signal to the pinhole 894.The pinhole 894 is configured to block a scattered portion in theoptical signal. In an example, the pinhole 893 has a diameter about 1000μm. The first acromat lens unit 895 is used to focus the optical signalonto the AOTF 896.

The AOTF 896 is an electrically tunable optical filter. In an example,the AOTF 896 includes an optically birefringent crystal, such astellurium dioxide (TeO₂). When the AOTF 896 receives an electricalsignal having a frequency, the AOTF 896 generates an acoustic wavehaving the frequency. Further, the acoustic wave is launched into thecrystal, and interacts with the optical signal in the crystal. As aresult, a portion of the optical signal is diffracted and exits thecrystal at an angle different from the rest of the optical signal. Theportion of the optical signal has a wavelength that satisfies a matchingcondition of the crystal with the frequency of the acoustic wave. In anexample, the portion of the optical signal satisfying the matchingcondition exits the crystal at about ±5°, and the rest of the opticalsignal exits the crystal without diffraction. When the frequency of theelectrical signal is changed, the AOTF 896 selectively diffracts anotherwavelength in the optical signal that satisfies the matching conditionwith the changed frequency.

The beam block 897 is coupled to the AOTF 896 to filter the opticalsignal to have the selected wavelength. More specifically, the beamblock 897 blocks the un-diffracted portion of the optical signal, andallows the diffracted portion of optical signal having the selectedwavelength to pass through. Then, the second acromat lens unit 898focuses filtered optical signal to the PMT 899.

The PMT 899 receives the filtered optical signal having the selectedwavelength, and generates an electrical signal, such as a currentsignal, a voltage signal, and the like, in response to the filteredoptical signal. In an example, an amplitude of the electrical signalcorresponds to the intensity of the filtered optical signal.

In an embodiment, multiple fluorescent labels are used for labeling DNAfragment. The multiple fluorescent labels can emit light of differentwavelengths. To detect the different wavelengths, a controller iscoupled to the AOTF 896. The controller adjusts a control signal tochange the frequency of the electrical signal input to the AOTF 896 inorder to select different wavelengths for the filtered optical signal.

FIG. 9 shows a block diagram of a signal processing path 900 accordingto an embodiment of the disclosure. The signal processing path includesan AOTF module 910, a PMT detector module 920, a phase sensitivedetector (PSD) module 930, a post processor module 940, a radiofrequency (RF) spectral tuning module 950, and a low-frequencymodulation module 960. These elements can be coupled together as shownin FIG. 9.

The RF spectral tuning module 950 includes circuits to generate anelectrical signal having a tunable radio frequency (RF). In anembodiment, the RF spectral tuning module 950 includes a controller anda synthesizer coupled together. The controller can be implemented as ageneral controller executing software instructions, or can beimplemented as application specific integrated circuit (ASIC). Thecontroller generates a control signal indicating a radio frequency, andprovides the control signal to the synthesizer. The synthesizergenerates the electrical signal having the radio frequency based on thecontrol signal. In an embodiment, the controller repetitively adjuststhe control signal corresponding to multiple radio frequencies. Thus,the electrical signal generated by the synthesizer repeats the multipleradio frequencies.

It is noted that the RF spectral tuning module 950 can include othercomponents to further process the electrical signal. In an example, theRF spectral tuning module 950 includes an RF amplifier to amplify theelectrical signal in the RF domain, and reduce harmonic frequencyportions in the electrical signal to clean the electrical signal. Then,the cleaned electrical signal is provided to the AOTF module 910.

The AOTF module 910 receives the electrical signal having the radiofrequency. Further, the AOTF module 910 imposes an acoustic wave havingthe radio frequency on a crystal, such as an optically birefringentcrystal. In an example, the AOTF module 910 includes a transducer, suchas a piezoelectric transducer, coupled with the crystal. The transducerconverts the electrical signal to the acoustic wave having the radiofrequency, and launches the acoustic wave into the crystal.

In addition, the AOTF module 910 receives an optical signal collectiveof excited fluorescence. The AOTF module 910 filters the optical signalto select a wavelength based on the electrical signal. The wavelengthsatisfies a matching condition of the AOTF module 910 with the radiofrequency of the electrical signal. More specifically, the acoustic wavehaving the radio frequency interacts with the optical signal on thecrystal. As a result, a portion of the optical signal is diffracted andexits the crystal at an angle different from the rest of the opticalsignal. The diffracted portion of the optical signal has a wavelengththat satisfies the matching condition of the AOTF module 910 with theradio frequency. In an example, the diffracted portion of the opticalsignal exits the crystal at about 5-7°, and the rest of the return beamexits the crystal without diffraction.

According to an embodiment of the disclosure, the AOTF module 910includes a beam-block to allow the diffracted portion of the opticalsignal to pass through, and block the un-diffracted portion of theoptical signal. The filtered optical signal is suitably directed to thePMT detector 920.

It is noted that when the electrical signal repeats the multiple radiofrequencies, the AOTF module 910 scans the optical signal for multiplewavelengths that respectively satisfy the matching condition of the AOTFmodule 910 with the multiple radio frequencies. Thus, the filteredoptical signal repetitively scans the multiple wavelengths.

The PMT detector 920 receives the filtered optical signal, and generatesan electrical signal corresponding the filtered optical signal. Morespecifically, the PMT detector 920 includes a tube that emits electronsin response to photons. The electrons can be suitably collected and usedto generate the electrical signal. Thus, an amplitude of the electricalsignal is proportional to the intensity of the filtered optical signal.The electrical signal is provided to the PSD module 930.

The PSD module 930 is coupled to the low frequency modulation module 960for reducing noises in the electrical signal. More specifically, thelow-frequency modulation module 960 provides a modulation signal to theAOTF module 910, and a reference signal to the PSD module 930. Themodulation signal and the reference signal have a relative low frequencycomparing to the radio frequencies generated by the RF spectral tuningmodule 950. The modulation signal is used by the AOTF module 910 tomodulate the filtered optical signal. Thus, the electrical signalgenerated in response to the filtered optical signal is modulated by therelative low frequency. The reference signal is used by the PSD module930 to demodulate the electrical signal to obtain a spectrally scannedelectrical signal. Thus, influences of noises originated in the PMTdetector module 920 can be reduced.

The spectrally scanned electrical signal can be suitably furtherprocessed, such as transferred, stored, digitalized, and the like. Inthe FIG. 9 example, the spectrally scanned electrical signal isprocessed by the post processor 940 to obtain spectrally separatedsignals 970. In an embodiment, the controller adjusts the control signalbased on a substantially constant time interval. The post processor 940can separate the spectrally scanned electrical signal based on thesubstantially constant interval to obtain the spectrally separatedsignal 970. The post processor 940 can be implemented as a generalprocessor executing software instructions for post processing, or can beimplemented as ASIC.

FIG. 10 shows a flow chart outlining a process example 1000 for acontroller, such as the controller 180, to control a detection moduleaccording to an embodiment of the disclosure. The process starts atS1001 and proceeds to S1010.

At S1010, the controller sends control signals to the detection moduleto initialize the detection module. For example, when a new samplecartridge having a microfluidic chip is installed in the DNA analyzer100, the controller 180 sends control signals to the detection module150 to initialize the detection module 150. In an example, the detectionmodule 150 aligns its objective lens with regard to a separation channelon the microfluidic chip. Thus, the objective lens can direct a laserbeam to a location along the separation channel, and can collectfluorescence excited by the laser beam. The process then proceeds toS1020.

At S1020, the controller determines multiple wavelengths for detection.In an example, the controller receives information about reagents usedin PCR and ILS added after PCR. Based on the information, the controllerdetermines types of fluorescent labels used to label DNA fragments, anddetermines the multiple wavelengths that can be emitted by thefluorescent labels. The controller may further determine radiofrequencies corresponding to the multiple wavelengths, and controlvalues to generate the radio frequencies. The controller may makedeterminations based on an AOTF module used to filter the fluorescence.For example, each wavelength for detection satisfies a matchingcondition of the AOTF module with one of the determined radiofrequencies. In an example, the controller includes a look-up table toassist the controller to make determinations. The process then proceedsto S1030.

At S1030, the controller provides a control signal to the detectionmodule. The control signal is indicative of a radio frequency. In anexample, the detection module includes a synthesizer. The synthesizergenerates an electrical signal having the radio frequency according tothe control signal. The electrical signal can be further processed, andprovided to the AOTF module. The AOTF module includes a transducer thatconverts the electrical signal into an acoustic wave and launches theacoustic wave into a crystal. The AOTF module also receives an opticalsignal. The optical signal includes fluorescence collected by theobjective lens from the separation channel. The optical signal interactsthe acoustic wave on the crystal. As a result, a portion of the opticalsignal having a wavelength satisfying the matching condition with theradio frequency can pass the AOTF module. The process then proceeds toS1040.

At S1040, the controller maintains the control signal for a timeduration. The time duration is enough for the AOTF module to settle andfilter the optical signal. The filtered optical signal is converted toan electrical signal by a photo-detector, such as PMT. The electricalsignal can be further processed, such as digitalized, stored, and thelike.

At S1050, the controller determines whether the detection process ends.When the detection process ends, the process proceeds to S1099 andterminates; otherwise, the process proceeds to S1060.

At S1060, the controller adjusts the control signal, and provides theadjusted control signal to the detection module. The adjusted controlsignal is indicative of another radio frequency that can be used toselect another wavelength. Similarly, the synthesizer generates theelectrical signal having the other radio frequency based on the adjustedcontrol signal. Then, the transducer in the AOTF module converts theelectrical signal into an acoustic wave and launches the acoustic waveinto the crystal. The acoustic wave interacts with the optical signal inthe crystal. As a result, a portion of the optical signal having theother wavelength can pass the AOTF module. Then, the process returns toS1040.

While the invention has been described in conjunction with the specificexemplary embodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, exemplary embodiments of the invention as set forthherein are intended to be illustrative, not limiting. There are changesthat may be made without departing from the spirit and scope of theinvention.

1. A DNA analyzer, comprising: an interface for coupling a microfluidicchip to the DNA analyzer, wherein the microfluidic chip includes: afirst domain configured for polymerase chain reaction (PCR)amplification of DNA fragments, the DNA fragments being tagged withfluorescent labels; and a second domain fluidically coupled to the firstdomain to receive the DNA fragments, the second domain having aseparation channel for electrophoretic separation of the DNA fragments;and a detection module optically coupled with the microfluidic chip thatincludes: a laser source configured to generate a laser beam; a set ofoptical elements configured to direct the laser beam to the separationchannel to excite the fluorescent labels to emit fluorescence while theDNA fragments migrate in the separation channel, and to collect theemitted fluorescence into an optical signal; a filter module configuredto filter the optical signal to allow a first portion of the opticalsignal having a first wavelength to pass; and a photo-detectorconfigured to generate an electrical detection signal in response to thefiltered optical signal.
 2. The DNA analyzer of claim 1, wherein thephoto-detector further comprises: a photo-multiplier tube configured togenerate the electrical detection signal in response to the filteredoptical signal.
 3. The DNA analyzer of claim 1, wherein the set ofoptical elements further comprise: an objective lens aligned with theseparation channel to direct the laser beam to the separation channeland to collect the emitted fluorescence from the separation channel. 4.The DNA analyzer of claim 3, further comprising a motor configured toalign the objective lens to the separation channel.
 5. The DNA analyzerof claim 1, wherein the filter module further comprises: anacousto-optic tunable filter (AOTF) configured to filter the opticalsignal to allow the first portion of the optical signal having the firstwavelength to pass based on an electrical tuning signal having a firsttuning frequency, the first wavelength satisfying a matching conditionof the AOTF with the first tuning frequency.
 6. The DNA analyzer ofclaim 5, further comprising: a controller configured to generate acontrol signal indicative of the first tuning frequency; and asynthesizer configured to generate the electrical tuning signal havingthe first tuning frequency based on the control signal.
 7. The DNAanalyzer of claim 6, wherein the controller adjusts the control signalto be indicative of a second tuning frequency, and that causes: thesynthesizer generates the electrical tuning signal having the secondtuning frequency based on the control signal; and the AOTF filters theoptical signal to allow a second portion of the optical signal having asecond wavelength to pass based on the electrical tuning signal, thesecond wavelength satisfying the matching condition of the AOTF with thesecond tuning frequency.
 8. The DNA analyzer of claim 5, furthercomprising: a modulation signal generator configured to generate amodulation signal having a modulation frequency, and a reference signalhaving the modulation frequency, the modulation signal being used by theAOTF to modulate the filtered optical signal; and a phase-sensitivedetector configured to receive the reference signal and the electricaldetection signal corresponding to the modulated filtered optical signal,and demodulate the electrical detection signal based on the referencesignal.
 9. The DNA analyzer of claim 1, further comprising: a pressuremodule configured to flow liquid in the microfluidic chip; a thermalmodule configured to induce thermal cycling at the first domain of themicrofluidic chip for the PCR amplification; a power module configuredto generate voltages to be applied to the second domain of themicrofluidic chip for the electrophoretic separation; and a controllermodule configured to control the pressure module, the thermal module,the power module, and the detection module according to a controlprocedure to act on the microfluidic chip for a single-chip DNAanalysis.
 10. A method of DNA analysis, comprising: selecting a firstwavelength corresponding to a first fluorescent label used to label DNAfragments during polymerase chain reaction (PCR) amplification in afirst domain of a microfluidic chip, the DNA fragments having beenfluidically directed from the first domain to a second domain of themicrofluidic chip having a separation channel for electrophoreticseparation; exciting at least the first fluorescent label to emitfluorescence in the second domain; and tuning a detection module todetect the emitted fluorescence having the first wavelength.
 11. Themethod of claim 10, wherein exciting the first fluorescent label to emitthe fluorescence in the second domain further comprises: generating alaser beam; and directing the laser beam to the separation channel toexcite the first fluorescent label to emit the fluorescence while theDNA fragments migrate in the separation channel.
 12. The method of claim11, further comprising: collecting the emitted fluorescence into anoptical signal.
 13. The method of claim 12, wherein tuning the detectionmodule to detect the emitted fluorescence having the first wavelength,further comprises: generating an electrical tuning signal having a firsttuning frequency; providing the electrical tuning signal to anacousto-optic tunable filter (AOTF) in the detection module to filterthe optical signal and pass a first portion of the optical signal havingthe first wavelength, the first wavelength satisfying a matchingcondition of the AOTF with the first tuning frequency; and detecting thefiltered optical signal.
 14. The method of claim 13, further comprising:selecting a second wavelength corresponding to a second fluorescentlabel used to label the DNA fragments during the (PCR) amplification inthe first domain; adjusting the electrical tuning signal to have asecond tuning frequency; and causing the AOTF to filter the opticalsignal and pass a second portion of the optical signal having the secondwavelength, the second wavelength satisfying the matching condition ofthe AOTF with the second tuning frequency.
 15. A DNA analyzer,comprising: an interface for coupling a microfluidic chip to the DNAanalyzer, wherein the microfluidic chip includes: a first domainconfigured for polymerase chain reaction (PCR) amplification of DNAfragments, the DNA fragments being tagged with fluorescent labels; and asecond domain fluidically coupled to the first domain to receive the DNAfragments, the second domain having a separation channel forelectrophoretic separation of the DNA fragments; a detection moduleoptically coupled with the microfluidic chip that includes: a lasersource configured to generate a laser beam; a passive optics moduleincluding passive units that are pre-configured to receive the laserbeam and transmit the laser beam; and an active optics module includingat least an active unit to focus the laser beam to the separationchannel to excite the fluorescent labels to emit fluorescence while theDNA fragments migrate in the separation channel, and to collect theemitted fluorescence from the separation channel into an optical signalfor return, wherein the passive optics module includes: a filter moduleconfigured to filter the optical signal to allow a first portion of theoptical signal having a first wavelength to pass; and a photo-detectorconfigured to generate an electrical detection signal in response tofiltered optical signal.
 16. The DNA analyzer of claim 15, wherein thephoto-detector further comprises: a photo-multiplier tube configured togenerate the electrical detection signal in response to the filteredoptical signal.
 17. The DNA analyzer of claim 15, wherein the activeoptics module further comprises: an objective lens aligned with theseparation channel to direct the laser beam to the separation channeland to collect the emitted fluorescence from the separation channel. 18.The DNA analyzer of claim 17, wherein the active optics module furthercomprises: a motor configured to align the objective lens to theseparation channel.
 19. The DNA analyzer of claim 15, wherein the filtermodule further comprises: an acousto-optic tunable filter (AOTF)configured to filter the optical signal to allow the first portion ofthe optical signal having the first wavelength to pass based on anelectrical tuning signal having a first tuning frequency, the firstwavelength satisfying a matching condition of the AOTF with the firsttuning frequency.
 20. The DNA analyzer of claim 19, further comprising:a controller configured to generate a control signal indicative of thefirst tuning frequency; and a synthesizer configured to generate theelectrical tuning signal having the first tuning frequency based on thecontrol signal.
 21. The DNA analyzer of claim 20, wherein the controlleradjusts the control signal to be indicative of a second tuningfrequency, and that causes: the synthesizer generates the electricaltuning signal having the second tuning frequency; and the AOTF filtersthe optical signal to allow a second portion of the optical signalhaving a second wavelength to pass based on the electrical tuningsignal, the second wavelength satisfying the matching condition of theAOTF with the second tuning frequency.
 22. The DNA analyzer of claim 19,further comprising: a modulation signal generator configured to generatea modulation signal having a modulation frequency, and a referencesignal having the modulation frequency, the modulation signal being usedby the AOTF to modulate the filtered optical signal; and aphase-sensitive detector configured to receive the reference signal andthe electrical detection signal corresponding to the modulated filteredoptical signal, and demodulate the electrical detection signal based onthe reference signal.
 23. The DNA analyzer of claim 15, furthercomprising: a pressure module configured to flow liquid in themicrofluidic chip; a thermal module configured to induce thermal cyclingat the first domain of the microfluidic chip for the PCR amplification;a power module configured to generate voltages to be applied to thesecond domain of the microfluidic chip for the electrophoreticseparation; and a controller module configured to control the pressuremodule, the thermal module, the power module, and the detection moduleaccording to a control procedure to act on the microfluidic chip for asingle-chip DNA analysis.